Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China.
Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China.
Nat Commun. 2023 May 29;14(1):3085. doi: 10.1038/s41467-023-38621-3.
Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.
基因的异构体可能有助于多种生物学功能。在耳蜗中，替代异构体的组合仍未被探索。我们整合了单细胞短读长和长读长 RNA 测序技术，鉴定了 236012 个转录本，其中 126612 个在 GENCODE 数据库中未被注释。然后，我们使用 RNA-seq、RT-PCR、Sanger 测序和基于 MS 的蛋白质组学方法分析和验证了未注释的转录本。为了说明鉴定剪接异构体的重要性，我们研究了参与内毛细胞（IHC）突触传递的关键蛋白 otoferlin。在删除经典 otoferlin 异构体后，鉴定出的短异构体能够支持正常的听力阈值，但 IHC 的持续胞吐作用降低，进一步揭示了 otoferlin 在之前未被充分解决的内吞膜回收中的作用。此外，我们发现 otoferlin 异构体与 IHC 功能和听觉表型有关。这项工作扩展了我们在异构体分辨率水平上对听觉功能的机制理解。
